This invention relates to the determination of the activated clotting time (ACT) of blood or plasma, specifically for patients receiving the drugs heparin and aprotinin.
Heparin is an anticoagulant drug used during surgeries requiring the use of extracorporial circuits, such as cardiopulmonary bypass (CPB). Heparin prevents blood clots from forming while blood is flowing through the circuit. In order to ensure sufficient anticoagulation, doctors must be able to measure heparin""s anticoagulant effect on blood. The anticoagulant effect of heparin is often managed using the activated clotting time (ACT) as determined by in vitro diagnostic tests. The ACT is prolonged after heparin administration. For example, without heparin, the ACT is generally about 120 seconds, while the ACT after heparin administration is typically beyond 480 seconds. An ACT of greater than 480 seconds is considered by many clinicians to be the minimum amount of heparin anticoagulation effect necessary for CPB surgery. The object of an ACT test is to indicate if adequate heparin has been administered to the patient in order to proceed with the surgical procedure without risk of forming blood clots.
Heparin is metabolized during surgery. Consequently, heparin must be administered both before and during CPB surgery in order to maintain a sufficient level of anticoagulation. Also, the anticoagulant effect of heparin varies from patient to patient. Therefore, different patients require different amounts of heparin, both before and during CPB surgery.
Several different tests are used throughout the world to manage heparin administration during CPB surgery. The most widely used tests for heparin management are the ACT, activated partial thromboplastin time (aPPT), and protamine titration of heparin. Of these tests, ACTs are the most common, least expensive, and most convenient way to manage heparin administration during a CPB surgical procedure. Heparin management protocols vary among medical institutions, but typically, when the ACT drops below a threshold time of typically 400 to 480 seconds, additional heparin is administered. ACT tests contain a contact activator to trigger clot formation. The two activators currently in general used for CPB surgery are diatomaceous earth (celite) and kaolin, a type of clay. Glass beads (silicon dioxide) are also used as a contact activator for ACT tests, although they are typically not used in CPB surgery because glass beads provide less contact activation than comparable amounts of celite or kaolin.
Several prior art instruments measure ACTs. Exemplary of these products are the Hemochron(copyright) 801 and Hemochron Jr. manufactured by International Technidyne Corporation, the Automated Coagulation Timer II and the Hepcon(copyright) Hemostasis Management System manufactured by Metronic HemoTec Inc., the Hemotec ACT manufactured by HemoTec, Inc., the Actalyke(copyright) manufactured by Array Medical, and the Sonoclot(copyright) Coagulation and Platelet Function Analyzer manufactured by Sienco, Inc. All of these instruments measure the time required to form a clot. Measurement time typically begins when the whole blood sample is mixed with an activator and ends when a clot is detected.
Aprotinin, manufactured under the name Trasylol(copyright) by Bayer Corporation, is a drug that has been shown to reduce blood loss after cardiopulmonary bypass (CPB) surey. Researchers do not completely understand the process or processes by which aprotinin achieves this reduction in bleeding. Aprotinin is a protease inhibitor neutralizing primarily plasmin and kallikrein. It also reduces glycoprotein loss in platelets, thereby preserving the platelets"" ability to aggregate after CPB. The net effects of aprotinin administration reduce the need for post-operative blood transfusions and re-exploratory surgery to stop bleeding. Aprotinin may have additional effects on hemostasis via other mechanisms.
An undesirable and potentially dangerous side effect of aprotinin is that it prolongs celite activated ACT results in the presence of heparin. For example, a celite ACT on a heparinized patient may be 480 seconds. The same patient with the same amount of heparin plus aprotinin may have an ACT substantially over 700 seconds. The celite ACT result is not a useful measurement for the heparin anticoagulant effect in the presence of aprotinin because the aprotinin prolongs the ACT result. Managing heparin with celite ACT tests is not effective because, in the presence of aprotinin, the ACT is substantially delayed, creating a risk of under heparinization and subsequent clotting during surgery. Also, any time wasted waiting for a 700-second ACT is a significant operating room cost and should be avoided when possible.
Kaolin activated ACTs have been investigated as a preferred alternative to celite activated ACTs for use with patients receiving both heparin and aprotinin. Numerous publications state that kaolin ACTs are not prolonged by aprotinin. These publications include J. S. Wang et al., xe2x80x9cMonitoring of Heparin-induced Anticoagulation with Kaolin-activated Clotting Time in Cardiac Surgical Patients Treated with Aprotinin,xe2x80x9d Anesthesiology, 77:1080-1084, December 1992, and W. Dietrich et al., xe2x80x9cInfluence of Hi-dose Aprotinin on Anticoagulation, Heparin Requirement, and Celite and Kaolin Activated Clotting Time in Heparin-pretreated Patients Undergoing Open-Heart Surgery,xe2x80x9d Anesthesiology, 88: 679-689, October 1995. The published research of kaolin activated ACTs appears to have been based on the assumption that all kaolin reagents perform similarly when used for ACTs. Each of these published studies investigated commercially available kaolin ACTs or custom kaolin ACTs using only one type of kaolin. The idea that the source or purity of kaolin may contribute to the stability of kaolin ACT results in the presence of heparin is never discussed.
The original development objective of the present inventors was to develop a kaolin ACT for use with the Sonoclot Analyzer in applications using aprotinin during CPB surgery. Based on published information, the initial assumption was that this development effort would be straightforward. The first kaolin formulations used USP grade kaolin, part number 5645 from Mallinckrodt Chemical, Inc., Kentucky, in a concentration that produced a desired heparin dose response to other commercial kaolin ACT tests. When this formulation was tested for aprotinin stability, an unexpected result was found; this kaolin ACT was found to perform similar to celite ACTs rather than published performance for kaolin ACTs. The ACT results were significantly prolonged by aprotinin when heparin was also present. Subsequent work by the present inventors showed that kaolin activated ACT tests are highly dependent on the source and purity of kaolin.
This research also exposed another unexpected concern; any alteration of kaolin ACT results due to aprotinin is highly patient variable. Using kaolin from ACT tests manufactured by International Technodyne, it was found that the ACT results on heparinized blood were unaffected by aprotinin for most samples. However, in approximately 10% of blood samples taken from the normal population, the ACT results on heparinized blood were substantially prolonged by aprotinin. This is a significant concern because the patients with heparinized blood samples that produce prolonged ACT results in the presence of aprotinin may receive significantly less heparin. These patients are at risk for under heparinization and subsequent clotting of the CPB circuit. Published performance data on kaolin ACTs reported only sample mean and standard deviations; this statistical analysis can miss individual patient variability that occurs only in a small percentage of the sample population. Kaolin ACT performance in the presence of aprotinin is described in more detail hereinafter.
The manufacturer of aprotinin recognizes that aprotinin may compromise ACT based heparin management. The aprotinin product insert recommends minimum ACTs while on CPB of 750 or 480 seconds for celite or kaolin ACTs, respectively. The aprotinin product insert also states that heparin may be monitored by either of two alternative ways separate from ACT testing. The first alternative method is to administer heparin on a fixed-dose regimen based on the patient""s weight and the duration of CPB, which is an older and less exact method for heparin management. The second alternative method is to measure the heparin level using a protamine titration. Neither of these methods is as easy, reliable, and convenient as measuring the ACT. In addition, the protamine titration method can increase the cost of the procedure by about $150.
In addition, measuring the level of heparin present in the blood is not as accurate an assessment of a patient""s anticoagulation state as measuring the ACT. Heparin is an indirect anticoagulant and is effective only through bonding with antithrombin, also called antithrombin III, a natural anticoagulant present in blood. Heparin bonded to antithrombin is a much stronger anticoagulant than antithrombin alone. Certain patients are insensitive or resistant to heparin because they have low levels of antithrombin. Therefore, simply measuring the level of heparin is not sufficient to determine the effective anticoagulative state of the patient. An ACT test, however, determines the overall anticoagulation state of the blood and can be useful in managing heparin anticoagulation therapy.
Array Medical manufactures an ACT test termed MAX-ACT that uses a mixture of standard materials, including kaolin, celite, and glass beads. This formulation is intended to maximize factor XII activation. This formulation was investigated and was found to perform substantially equivalent to commercially available kaolin ACT formulations.
An ideal ACT test should have a dose response relationship solely influenced by heparin. Higher ACT results should indicate higher concentrations of circulating heparin. The absence or presence of aprotinin should not alter the ACT result. Current ACT formulations fall short of ideal ACT performance when aprotinin is used. For patients receiving both heparin and aprotinin, heparin prolongs the ACT result, and aprotinin may further prolong the test result. Consequently, in the presence of aprotinin, current ACT test results do not indicate the level of heparin anticoagulant effect and may expose the patient to a risk of inadequate and unsafe heparin administration. Celite tests are significantly prolonged by a combination of aprotinin and heparin in comparison to heparin alone. Kaolin ACT tests perform inconsistently in the presence of aprotinin. Some kaolin formulations perform similarly to celite ACT tests, and results are consistently prolonged in the presence of aprotinin and heparin in comparison to heparin alone. Other kaolin-activated ACT tests, including the kaolin used by International Technidyne Corporation, produce inconsistent results in the presence of both heparin and aprotinin. In most blood samples, the aprotinin has little effect on the ACT, while in others the ACT is substantially prolonged by aprotinin. With the kaolin ACT formulation used by International Technidyne Corporation, a prolonged ACT test result, for a blood sample that happens to be insensitive to aprotinin using this test formulation, would be due exclusively to the heparin, while in another blood sample that is sensitive to aprotinin, the ACT test result may be prolonged due to the combined effects of both the heparin and the aprotinin. Consequently, currently manufactured kaolin ACT test results are highly inaccurate in measuring the heparin anticoagulation effect for individual patients that show strong sensitivity to aprotinin in the presence of heparin when using a kaolin ACT. An ACT formulation that is less affected by aprotinin for a larger percentage of patients will improve anticoagulant safety in CPB surgery.
It would therefore be advantageous to provide an improved activated clotting time formulation that provides consistent response to the anticoagulant heparin regardless of the absence or presence of the drug aprotinin and a method for quantifying the aprotinin sensitivity of different activation formulations to evaluate aprotinin sensitivity performance.
One feature of the present invention is the provision of an in vitro diagnostic test useful for characterizing the anticoagulation effect of heparin that is not altered by the drug aprotinin.
Another feature of the present invention is the provision of an in vitro diagnostic test that also produces test results similar to current widely used tests for characterizing the anticoagulation effect of heparin.
Another feature of the present invention is the provision of an in vitro diagnostic test that further provides consistently equivalent results in the presence of heparin regardless of the absence or presence of aprotinin for a wide range of individuals rather than just average equivalence for a sample population.
Yet another feature of the present invention is the provision of a method to evaluate the performance of different activation formulations for an in vitro diagnostic test that is to be used to characterize the anticoagulation effect of heparin in the presence or absence of aprotinin that accounts for both average performance for a sample population and individual patient variability to different activation formulations so that accurate heparin anticoagulation management can be achieved for a large percentage of individual patients.